Pathogen detection by simultaneous size/fluorescence measurement

ABSTRACT

A method and apparatus for detecting pathogens and particles in a fluid in which particle size and intrinsic fluorescence of a simple particle is determined.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Non-Provisional Applicationhaving Ser. No. 11/768,103, filed on Jun. 25, 2007, now abandoned whichin turn claims priority to U.S. provisional application having Ser. No.60/805,962, filed on Jun. 27, 2006. The contents these applications areincorporated by reference herein in their entirety, for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to a system and method fordetecting airborne or waterborne particles, and more particularly to asystem and method for detecting airborne or waterborne particles andclassifying the detected particles. The invention has particular utilityin detecting and classifying allergens and biological warfare agent, andwill be described in connection with such utility, although otherutilities are contemplated.

BACKGROUND OF THE INVENTION

An urban terrorist attack involving release of biological warfare agentssuch as bacillus anthracis (anthrax) is presently a realistic concern.Weaponized anthrax spores are extremely dangerous because they can gainpassage into the human lungs. A lethal inhalation dose of anthrax sporesfor humans, LD50 (lethal dose sufficient to kill 50% of the personsexposed) is estimated to be 2,500 to 50,000 spores (see T. V. Inglesby,et al., “anthrax as a Biological Weapon”, JAMA, vol. 2801, page 1735,1999). Some other potential weaponized bio-agents are yersinia pestis(plaque), clostridium botulinum (botulism), and francisella tularensis.In view of this potential threat, there is currently a need for an earlywarning system to detect such an attack. In the pharmaceutical,healthcare and food industries, a real time detector of environmentalmicrobial level is useful for public health, quality control andregulatory purposes. For example, parental drug manufacturers arerequired to monitor the microbial levels in their aseptic clean rooms.In these applications, an instrument which can detect microbes in theenvironment instantaneously will be a useful tool and have advantagesover conventional nutrient plate culture methods which requires days formicrobes to grow and to be detected.

Particle size measurement and ultraviolet (UV) induced fluorescencedetection have been used to detect the presence of biological substancesin the air. There exist various patents describing using thesetechniques as early warning sensors for bio-terrorist attack release ofweaponized bio-agents. Among these devices are Biological Agent WarningSensor (BAWS) developed by MIT Lincoln Laboratory, fluorescencebiological particle detection system of Ho (Jim yew-Wah Ho, U.S. Pat.Nos. 5,701,012; 5,895,922; 6,831,279); FLAPS and UV-APS by TSI ofMinnesota (Peter P. Hairston; and Frederick R. Quant; U.S. Pat. No.5,999,250), and a fluorescence sensor by Silcott (U.S. Pat. No.6,885,440).

A proposed bio-sensor based on laser-induced fluorescence using a pulsedUV laser is described by T. H. Jeys, et al., Proc. IRIS Active Systems,vol. 1, p.235, 1998. This is capable of detecting an aerosolconcentration of five particles per liter of air, but involves expensiveand delicate instruments. Other particle counters are manufactured byMet One Instrument, Inc, of Grants Pass, Oreg., Particle MeasurementSystems, Inc., of Boulder, Colo., and Terra Universal Corp., of Anaheim,Calif.

Various detectors have been designed to detect airborne allergenparticles and provide warning to sensitive individuals when the numberof particles within an air sample exceeds a predetermined minimum value.These are described in U.S. Pat. Nos. 5,646,597, 5,969,622, 5,986,555,6,008,729, 6,087,947, and 7,053,783, all to Hamburger et al. Thesedetectors all involve direction of a light beam through a sample ofenvironmental air such that part of the beam will be scattered by anyparticles in the air, a beam blocking device for transmitting only lightscattered in a predetermined angular range corresponding to thepredetermined allergen size range, and a detector for detecting thetransmitted light.

SUMMARY OF THE INVENTION

For the purpose of detection of microbes in air or water, it is ofimportance to devise an effective system to measure both particle sizeand fluorescence generated intrinsically by the microbes. The presentinvention provides a sensor system which is capable of simultaneouslymeasuring particle size and detecting the presence of intrinsicfluorescence from metabolites and other bio-molecules, on aparticle-by-particle basis. The advantages of this detection scheme overthe prior art are several. For one it provides a deterministic particlemeasurement methodology for characterizing particles rather than relyingon statistical models employed in prior art for particlecharacterization. The deterministic measurement methodology enables moredefinitive assignment of particle characters than the prior art and lessreliance on statistical models. It also reduces the possibility of falsepositives in microbial detection, for example, pollen (larger sizes thanmicrobes) and smoke particles (smaller sizes than microbes) can beexcluded from detection. And, it allows detailed analyses of datacollected on each individual particle for characterizing the particle,such as intensity of fluorescence signal from a particle as a functionof its cross-sectional area or volume, for the purpose of determiningthe biological status of the particles.

The current invention comprises three main components: (1) a firstoptical system for measuring an individual particle size; (2) a secondoptical system to detect a UV laser-induced intrinsic fluorescencesignal from an individual particle; and (3) a data recording format forassigning both particle size and fluorescence shy to an individualparticle, and computer readable program code for differentiatingmicrobes from non-microbes (e.g. inert dust particles).

The optical assembly of the present invention has two opticalsub-assemblies: (a) an optical setup to measure the particle size. As anexample, the preferred embodiment of the current invention uses thewell-known and often used Mie scattering detection scheme, but appliesit in a novel way, enabling the system to make highly accuratemeasurements of airborne particles with size ranges from 0.5 microns to20 microns. This capability to make fine distinctions in size isimportant in order to determine the class of microbe, because differentclasses of microbes have different size ranges; (b) simultaneous to theparticle size measurement, an optical apparatus is used to measure thefluorescence level from the particle being interrogated. As an example,the preferred embodiment of the current invention uses an ellipticalminor which is positioned to collected fluorescence emission from thesame particle as it is being measured for size.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will be seenfrom the following detailed description, taken in conjunction with theaccompanying drawings, wherein:

FIG. 1 is a plot showing particle size ranges of several airborne inertand microbial particulates;

FIG. 2( a) is a histogram representation of simultaneous measurements ofparticle size and fluorescence showing particle distribution formicrobe-free air;

FIG. 2( b) is a histogram showing simultaneous measurements of particlesize and fluorescence for air containing Baker's yeast powder;

FIG. 3 is a histogram representation of simultaneous measurements of 7micron size fluorescent dye doped particles and fluorescence;

FIG. 4 is a schematic diagram of an optical system accordance with thepresent invention, for performing simultaneous measurements of particlesize and fluorescence; and

FIG. 5 is a block diagram of the optical system of FIG. 4.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 4 is a schematic representation of an optical system for a fluidparticle detector system according to a first exemplary embodiment ofthe invention. This first exemplary embodiment of the system isdesigned, for example to detect airborne or waterborne bio-terroristagents deliberately released by terrorists or others, but also may beused in civilian applications to detect harmful levels of other airborneor waterborne particles which may exist naturally such as mold orbacteria, or which may have been accidentally, inadvertently, naturally,or deliberately related, or for other industrial applications such asthe food and pharmaceutical manufacturing industries, as well as cleanroom applications.

The term “fluid borne particles” as used herein means both airborneparticles and waterborne particles.

The term “pathogen” as used herein refers to any airborne or waterborneparticles, biological agent, or toxin, which could potentially harm oreven kill humans exposed to such particles if present in the air orwater in sufficient quantities.

The term “biological agent” is defined as any microorganism, pathogen,or infectious substance, toxin, biological toxin, or any naturallyoccurring, bioengineered or synthesized component of any suchmicroorganism, pathogen, or infectious substance, whatever As origin ormethod of production. Such biological agents include, for example,biological toxins, bacteria, viruses, rickettsiae, spores, fungi, andprotozoa, as well as others known in the art.

“Biological toxins” are poisonous substances produced or derived fromplants, animals or microorganisms, but also can be produced or alteredby chemical means. A toxin, however, generally develops naturally in ahost organism (i.e., saxitoxin is produced by marine algae), butgenetically altered and/or synthetically manufactured toxins have beenproduced in a laboratory environment. Compared with microorganisms,toxins have a relatively simple biochemical composition and are not ableto reproduce themselves. In many aspects, they are comparable tochemical agents. Such biological toxins are, for example, botulinum andtetanus toxins, staphylococcal enterotoxin B, tricothocene mycotoxins,ricin, saxitoxin, Shiga and Shiga-like toxins, dendrotoxins, erabutoxinb, as well as other known toxins.

The detector system of the present invention is designed to detectairborne or waterborne particles and produce outputs indicating, forinstance, the number of particles of each size within the range, whichis detected in a sample, and indicate whether the particles are biologicor non-biologic. The system also may produce an signal or other responseif the number of particles exceeds a predetermined value above a normalbackground level, and/or biological organisms or biological agents andpotentially dangerous.

FIG. 4 is a representation of system 10 for a fluid particle detectorsystem according to an exemplary embodiment of the invention. As shownin FIG. 4, the system 10 includes an UV light excitation source 12 suchas a laser providing a beam of electromagnetic radiation 14 have an UVlight source wavelength. The UV light source is selected to have awavelength capable of exciting intrinsic fluorescence from metabolitesinside microbes. By way of example, the excitation source 12 preferablyoperates in a wavelength of about 270 nm to about 410 nm, preferablyabout 350 nm to about 410 nm. A wavelength of about 270 nm to about 410nm is chosen based on the premise that microbes comprise three primarymetabolites: tryptophan, which normally fluoresces at about 270 nm witha range of about 220 nm-about 300 nm; nicotinamide adenine dinucleotide(NADH) which normally fluoresces at about 340 nm (range about 320nm-about 420 nm); and riboflavin which normally fluoresces at about 400nm (range about 320 nm-about 420 nm). Preferably, however, theexcitation source 12 has a wavelength of about 350 to about 410 nm. Thiswavelength ensures excitation of two of the three aforesaid primarymetabolites, NADH, and riboflavin in bio-agents, but excludes excitationof interferences such as from diesel engine exhaust and other inertparticles such as dust or baby powder. Thus, in a preferred embodimentthe present invention makes a judicial selection of wavelength range ofthe excitation source 12, which retains the ability of excitingfluorescence from NADH and riboflavin (foregoing the ability to excitetryptophan) while excluding the excitation of interferents such asdiesel engine exhaust. This step is taken to reduce false alarmsgenerated by diesel exhaust (which can be excited by short UVwavelengths such as 266 nm light.

In the system 10 illustrated in FIG. 4, environmental air (or a liquidsample) is drawn into the system through a nozzle 16 for particlesampling. Nozzle 16 has an opening 18 in its middle section to allow thelaser beam to pass through the particle stream. Directly downstreamfront the laser beam is a Mie scattering particle-size detector 20. Miescattering particle-size detector 20 includes a beam blocker lens 22, acollimator lens 24 and a condenser lens 26 for focusing a portion of thelight beam 14 onto a particle detector 28.

Off axis from the laser beam 14, an elliptical mirror 30 is placed atthe particle-sampling region in such a way that the intersection of theincoming particle stream and the laser beam is at one of the two foci ofthe ellipsoid, while a fluorescence detector 32 (in this case aphoto-multiplier tube) occupies the other focus. This design utilizesthe fact that a point source of light emanating from one of the two fociof an ellipsoid will be focused onto the other. In this optical design,the elliptical mirror 30 concentrates the fluorescence signal frommicrobe and focus it onto the fluorescence detector 32. An opticalfilter 34 is placed in front of the fluorescence detector to block thescattered UV light and pass the induced fluorescence.

The beam blocker lens 22 is designed to reflect non-scattered elementsof the laser beam 14, and may have a material, such as vinyl, attached afront surface to reflect the non-scattered elements of the beam ofelectromagnetic radiation. Other features and considerations for thebeam blocker lens 22 are disclosed in some of the earlier US patents toHamburger et al. listed above, and in PCT Application Serial No.PCT/US2006027638, incorporated herein by reference.

The particle detector 20 may comprise, for example, a photodiode forsizing the particles, e.g. as described in the earlier US patent toHamburger et al., listed above, and incorporated herein by reference Thepresent invention's use of Mie scattering also facilitates the placementof optical components for the detection of UV light illumination toconcurrently examine individual particles for the presence of themetabolites NADH, riboflavin and other bio-molecules, which arenecessary intermediates for metabolism of living organisms, andtherefore exist in microbes such as bacteria and fungi. If thesechemical compounds exist in a bio-aerosol, they are excited by the UVphoton energy and subsequently emit auto-fluorescence light which may bedetected by an instrument based on the detection scheme outlined above.While this detection scheme is not capable of identifying the genus orspecies of microbes, and viruses may be too small and lack themetabolism for detection, this detection scheme's ability tosimultaneously and for each particle determine the size of the particleand if it is biologic or inert indicates to the user the presence Orabsence of microbial contamination.

Referring to FIG. 5, the functionality of the simultaneous particlesizing and fluorescence measurement scheme of the present invention isdepicted in the graphic presentation of the measurement results fromsuch as an instrument. The principle of operation is as follows: aninstrument continuously monitors the environmental air (or liquid) tomeasure the size of each individual airborne particle in real time andto concurrently determine whether that particle emits fluorescence ornot. A threshold is set for the fluorescence signal. If the fluorescencesignal is below the set level, the particle is marked inert. Thisfluorescence signal threshold can be fluorescence signal intensity,fluorescence intensity as a function of particle cross-sectional area ora function of particle volume. If the fluorescence signal thresholdexceeds the set level, the particle is marked biological. The combineddata of particle size and fluorescence signal strength will determinethe presence or absence of microbes on a particle-by-particle basis.FIGS. 2( a) and 2(b) illustrate the functionality of a detector inaccordance with the present invention. They show the environmentalairborne particle data measured by using this detection scheme. In eachgraph, the upper part depicts in logarithmic scale the particle sizehistogram of particle concentration (#/liter of air) versus particlesize (from 1 micron to 13 microns); solid bars represent inert particleswhereas striped bars indicate the presence of microbes. The lower partof the graph is a real-time snap shot of the particles detected within 1second: each spike represents one single particle and its heightcorresponds to the particle size. In FIG. 2( a), the test was done forclean air, so there were only inert particles, free from microbes in asecond test, Baker's yeast powder (Saecharernyces cerevisiae) wasreleased into the air. The presence of the microbe was detected andshown by the striped bars in the histogram in FIG. 2( b).

FIG. 3 shows the data set obtained when 7 microns fluorescent dye dopedplastic beads were disseminated into a detector capable of simultaneousparticle size and fluorescence measurement scheme. The striped bars showthe presence of fluorescence in those particles with a distribution inthe 7 microns size range.

It should be emphasized that the above-described embodiments of thepresent invention, particularly, any “preferred” embodiments, are merelypossible examples of implementations, merely set forth for a clearunderstanding of the principles of the invention. Many variations andmodifications may be made to the above-described embodiments of theinvention without departing substantially from the spirit and principlesof the invention. All such modifications and variations are intended tobe included herein within the scope of this disclosure and the presentinvention and, protected by the following claims.

The invention claimed is:
 1. A particle detector system, comprising: asample cell; a light source on one side of a sample cell for sending afocused beam of light along a path to and through the sample, thedirection of the beam of light through the sample cell defining an axis,whereby portions of the beam of light are scattered at various angles byparticles of various sizes present in the sample area, and anunscattered portion of the beam of light remains unscattered; a beamblocking device on an opposite side of the sample cell along the axisfor blocking at least the unscattered portion of the beam of light andconfigured to limit a range of particles measured, such that lightscattered from particles of a predetermined size range proceeds past thebeam blocking device along a light path; a first detector positioned inthe light path after the beam blocking device along the axis fordetecting a portion of forward scattered light, and producing an outputincluding information on the size of a single particle in the light pathwithin a predetermined size range; a second detector positioned off theaxis from the beam of light for detecting intrinsic fluorescence fromsaid same single particle.
 2. The system of claim 1, wherein anelliptical mirror is located in a particle sampling region such that anintersection of the incoming particle stream and the light beam are atone foci of the ellipsoid, and the second detector is at the other foci.3. The system of claim 1, further comprising an alarm unit for providinga warning signal when a particle within a predetermined size range isdetected which also fluoresces.
 4. The system of claim 1, wherein thelight source emits ultraviolet radiation.
 5. The system of claim 1,wherein the light source comprises a LED.
 6. The system of claim 5,further comprising a collimator lens optically positioned between thelight source and the first detector.
 7. The system of claim 1, furthercomprising a processing unit for processing particle size distributionand particle fluorescence at a give time, and displaying a histogram ofthe particle on an output device.
 8. The system of claim 1, wherein thefirst detector comprise a photdiode.
 9. The system of claim 1, whereinthe sample cell comprises an air sample cell.
 10. The system of claim 1,wherein the sample cell comprises a water sample cell.
 11. The system ofclaim 1, further comprising computer readable program code forintegrating detected particle size and detected intrinsic fluorescence.